Browsing All Christie Publications by Subjects
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The expression of full length Gp91-phox protein is associated with reduced amphotropic retroviral production.BACKGROUND AND OBJECTIVE: As a single gene defect in mature bone marrow cells, chronic granulomatous disease (X-CGD) represents a disorder which may be amenable to gene therapy by the transfer of the missing subunit into hemopoietic stem cells. In the majority of cases lack of Gp91-phox causes the disease. So far, studies involving transfer of Gp91-phox cDNA, including a phase I clinical trial, have yielded disappointing results. Most often, low titers of virus have been reported. In the present study we investigated the possible reasons for low titer amphotropic viral production. DESIGN AND METHODS: To investigate the effect of Gp91 cDNA on the efficiency of retroviral production from the packaging cell line, GP+envAm12, we constructed vectors containing either the native cDNA, truncated versions of the cDNA or a mutated form (LATG) in which the natural translational start codon was changed to a stop codon. Following derivation of clonal packaging cell lines, these were assessed for viral titer by RNA slot blot and analyzed by non-parametrical statistical analysis (Whitney-Mann U-test). RESULTS: An improvement in viral titer of just over two-fold was found in packaging cells containing the start-codon mutant of Gp91 and no evidence of truncated viral RNA was seen in these cells. Further analysis revealed the presence of rearranged forms of the provirus in Gp91-expressing cells, and the production of truncated, unpackaged viral RNA. Protein analysis revealed that LATG-transduced cells did not express full-length Gp91-phox, whereas those containing the wild-type cDNA did. However, a truncated protein was seen in ATG-transduced cells which was also present in wild type cells. No evidence for the presence of a negative transcriptional regulatory element was found from studies with the deletion mutants. INTERPRETATION AND CONCLUSIONS: A statistically significant effect of protein production on the production of virus from Gp91-expressing cells was found. Our data point to a need to restrict expression of the Gp91-phox protein and its derivatives in order to enhance retroviral production and suggest that improvements in current vectors for CGD gene therapy may need to include controlled, directed expression only in mature neutrophils.
Specific heparan sulfate saccharides mediate the activity of basic fibroblast growth factor.In a previous study, we showed that heparitinase releases a 14-saccharide sequence (Oligo-H) from heparan sulfate (HS) with the structure delta GlcUA beta 1,4GlcNSO3-alpha 1,4[IdceA(2S)alpha 1,4GlcNSO3]5 alpha 1,4IdceA alpha 1,4GlcNAc (where IdceA(2S) represents iduronic acid 2-sulfate), which binds to basic fibroblast growth factor (bFGF) with high affinity (Turnbull, J. E., Fernig, D., Ke, Y., Wilkinson, M. C. & Gallagher, J. T. (1992) J. Biol. Chem. 267, 10337-10341). This paper describes further work on the binding properties of HS saccharides and their capacity to mediate bFGF activity in a mitogenesis assay in which responsiveness is dependent on the addition of HS or heparin. Saccharides prepared by heparinase or nitrous acid digestion and heparitinase-resistant fragments five disaccharide units (degree of polymerization (dp) = 10) or less in size were unable to activate bFGF. However, heparitinase-resistant saccharides of dp12-16 were active in the assay; the dp14 and dp16 fractions were equivalent in activity to heparin and more active than the parent HS. Saccharides of the same size and basic structure as the active fractions (> or = dp12) bound to bFGF with high relative affinity. Active saccharides were composed mainly of N-sulfated disaccharides, the predominant unit being IdceA(2S)-GlcNSO3. This was enriched at least 5-fold in the active saccharides by comparison with the original HS. In addition, the dp12 and dp14 active fractions had a notably low content of trisulfated disaccharides (IdceA(2S)-GlcNSO3(6S)) (where GlcNSO3(6S) represents N-sulfated glucosamine 6-sulfate), which are the major repeat units of heparin. The data show that sequences similar in size and basic structure to Oligo-H can mediate the mitogenic activity of bFGF. Overall, the results provide further evidence that specific HS sequences are generated biosynthetically in order to fulfill particular biological functions such as activation of bFGF.
Uptake of alpha-(L)-iduronidase produced by retrovirally transduced fibroblasts into neuronal and glial cells in vitro.The uptake of recombinant alpha-(L)-iduronidase into glial and neuronal cells, produced by retrovirally transduced NIH3T3 fibroblasts, was studied. We demonstrate that: (1) neuronal and glial cells take up alpha-(L)-iduronidase released into the medium by retrovirally transduced fibroblasts expressing high levels of alpha-(L)-iduronidase; (2) both glial and neuronal cells express the cation independent mannose-6-phosphate receptor responsible for lysosomal enzyme uptake; and (3) uptake of the lysosomal enzyme can be blocked by excess free mannose-6-phosphate, but not glucose-6-phosphate. Thus, various brain cells take up alpha-(L)-iduronidase, possibly through a cation independent mannose-6-phosphate receptor mediated pathway, and this uptake is higher in actively dividing or immature brain cells. Consequently, (1) neuronal metabolism ought to be capable of cross correction by enzyme provided by genetically engineered and transplanted cells provided by bone marrow transplantation (BMT); (2) that BMT could have a more beneficial effect on neurological function if performed as early as possible; and (3) given that the uptake mechanism of glial cells has a higher capacity, it might be easier to target diseases like the leukodystrophies in which lysosomal enzymes are needed in glial cells, compared to diseases where lysosomal enzymes ought to be delivered into neurons.